| Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck cancers. In China, the northeast part is the high incidence region of laryngeal carcinoma. The incidence of LSCCs has been rising in China over past decades. Laryngeal cancer cells are not sensitive to radiotherapy and chemotherapy, so surgery is the primary method in treating LSCC patients. However, surgery could bring different degrees of damage to LSCC patients. At presence, a common question in the diagnosis and treatment process is the hidden incidence of laryngeal carcinoma, which means patients diagnosed are often found in the middle or late stage. Therefore, it is urgent requirement to find a new way to detect or diagnose LSCC in early stage.Malignant tumors are considered as genetic and epigenetic diseases. Genes play an important role in tumorignesis. The alteration of gene seqence could resullt in the abnormal expressions of the related gene usually leading to the abnormal phenotype. However, gene expression is also controlled by epigenetic events. In recent years, gene expressions regulated by epigenetic mechanisms have become a hotspot in studying human diseases. DNA methylation, histone modifications and RNA interference are involved in epigenetic processes, among which DNA methylation is highly concerned. Epigenetic mechanisms are closely related to the occurrence and development of many diseases including cancers. DNA methylation is a reversible process of modification. DNA methylation inhibitors, such as 5'-aza-deoxycytidine (5'-Aza-CdR) which can inhibit DNA methyltransferase activity and change the methylation status of tumor-related genes so that gene function is restored or enhanced, become prospective reagents in treating tumor cells. By using 2-DE (two dimensional gel electrophoresis) and mass spectrometry (MS) technologies combining bioinformatics resource, we previously obtained 5-AZA-CdR-associated protein profiles and some important genes such as S100A4 in laryngeal carcinoma. Human S100A4 gene, which is located on 1q21, codes a 11.7kDa peptide with.101 amino acids. S100A4 participates in biological processes such as cytoskeleton formation, cell shape and signal transduction. The abnormal expression levels of S100A4 were found in esophageal cancer, stomach cancer, colon cancer and melanoma, but the mechanism of S100A4 involved in tumorigenesis is still unclear, and the associated data of S100A4 with laryngeal cancer have not been reported. This study aims to discover the function as well as the possible mechanism of S100A4 gene in the genesis and development of LSCC.Materials and MethodsClinical laryngeal carcinoma specimens and laryngeal squamous cell line Hep2 were used in this study. S100A4 gene expression was detected by using RT-PCR and Western blot. The methylation status of S100A4 gene was detected by using mathylation-specific PCR (MSP). Specific siRNA for S100A4 gene (S100A4-siRNA) designed and synthesized in vitro was transfected into Hep2 cells by TransMessenger Transfection reagent. The inhibitory effect of S100A4-siRNA on S100A4 gene in Hep2 cell line was evaluated by RT-PCR and Western Blot. Biological changes of Hep2 cells tranfected by S100A4-siRNA were detected by MTT, flow cytometry, transwell, RT-PCR and Western Blot. The wild-type and mutant-type fluorescent report luciferase expression vectors including the specifically methylated sites of S100A4 promoter were constructed to detect and analyse the transcriptional regulation role of S100A4 gene. The bioinformatics prediction software was used to predict the transcription factors which may bind S100A4. The predicted transcription factors were tested by using EMSA and CHIP. 1. RT-PCR and Western blot results showed that both S100A4 mRNA and protein levels were significantlly higher in human laryngeal carcinoma and metastatic lymph nodes than those in the adjacent control tissues.2. MSP results showed that hypomethylation within S100A4 promoter and first intron was found and related to the overexpression of S100A4 in human laryngeal carcinoma.3. Influence of S100A4-siRNA on the expression of S100A4 mRNA and protein:In day 5 of S100A4-siRNA transfection to Hep2 cells, S100A4 mRNA level was significantly lower than that in the control group and in day 7 of S100A4-siRNA transfection to Hep2 cells, S100A4 protein level was significantly lower than that in the control group, which indicated that S100A4 expression was inhibited by S100A4-siRNA.4. Influence of S100A4-siRNA on biological characteristics of laryngeal carcinoma cells Hep2:Compared to the control group, the abilities of proliferation and invasiveness were significantly lower, and apoptotic cells were significantly higher in Hep2 cells tranfected by S100A4-siRNA.5. Predictions of S100A4 regulatory site binding to transcription factors:The searching results from P-MATCH software displayed that transcription factor of c-Myb, c/Ebp, AP2, and MSX-1 might bind the corresponding sites of S100A4 regulatory region.6. Luciferase results showed that the methylation status of S100A4 regulatory region could change the promoter activities of S100A4.7. EMS A results showed that c-Myb and c/Ebp except AP2 and MSX-1 specifically bound S100A4 in vitro.8. ChIP results proved the specific binding of c-Myb and c/Ebp to S100A4 in vivo.Conclusion1. Overexpression of S100A4 in human laryngeal carcinoma and metastatic lymph nodes participates in the genesis and development of LSCC. 2. The methylation status of S100A4 promoter and first intron contributes to the overexpression of S100A4 in LSCC.3. S100A4 can promote the growth and invasiveness and inhibit the apoptosis of laryngeal cancer cells, which indicates S100A4 is an important oncogene associated with LSCC.4. C-Myb and c/Ebp could bind S100A4 gene specifically, and the C-Myb methylation status in binding sites of S100A4 gene affects the expression of S100A4 gene...
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